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why cant get the band? - (Aug/24/2011 )

Hi folks:

I badly need your guys help.

I am doing a reverse transcriptase PCR to amplify a gene from cDNA.
this gene expresses very well in the form of protein, which shows a very strong band by western blot.
however, after amplification by pcr, i barely detected the band. I use a plasmid of this gene as templet to amplify with the same primers, it shows very strong band as a positive control, indicating the system works, but why i cant get the band from cDNA amplification product, given the fact that it is a quite abundant expressed protein? can anyone help me to tackle this issue, i appreciate much.

best

dan

-dandan-

Most likely your RNA is degraded or your RT reaction was not successful. Are you be able to amplify another gene from the cDNA template, for example, a housekeeping gene? Have you checked your RNA quality by running an agarose gel?

-pcrman-

thanks Moderator, yeh the GAPDH looks perfect, :-(((( cant figure out why

-dandan-

please be aware that because housekeeping gene expression is very abundant, and can be amplified even there is RNA degradation. While other low expressing genes may not.

-pcrman-

i see. i have just checked the concentration of the RNA product, it is still fine.
meanwhile i will check another gene beside housekeeping gene as well. how to check if rna is degraded? thanks

-dandan-